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Summary of the impact

Research by the School of Pharmacy has underpinned the development of
fluorescent ligand probes that have opened-up new pathways in drug
discovery. These ligands have been commercialised through the formation of
the spin-out company CellAura Technologies Ltd, and have been made
globally available through a number of distributer agreements. Customers
include pharmaceutical companies (e.g. Pfizer, AstraZeneca), drug
discovery biotechs (e.g. Addex, Heptares) and drug discovery technology
providers (e.g. CisBio). These ligands provide alternatives to the use of
radio-ligands, giving more informative and safer solutions for industrial
drug discovery. This has, for example, enabled: a new direction in G
protein-coupled receptor research at Novartis Pharmaceuticals UK Ltd;
validation of Promega Corporation's new drug-binding assay; and superior
performance in the establishment of cell lines at inSCREENex GmbH.

Underpinning research

G protein-coupled receptors (GPCRs) are key drug targets in the
pharmaceutical industry. They control or influence a diverse range of
physiological functions and are the target for approximately 45% of all
currently licensed therapeutic drugs. The ability to measure the binding
of GPCR ligands and their resultant responses is therefore a key part of
the drug discovery process. Conventional methods using radioactively
labelled ligands (radio-ligand binding) are not suitable for the new
fluorescence based high-throughput techniques being used for drug
discovery. Radio-ligands also have significant health, safety and
environmental implications. Fluorescent ligand technologies offer an
updated approach with much improved detection limits compared to
radio-ligands with the ability to determine receptor-ligand interactions
at the single cell and single molecule level. This new approach
encapsulates the synthesis of fluorescently tagged small molecule
compounds that bind specifically and with high affinity to GPCRs, and
other membrane receptors, to allow biological properties of the
receptor-ligand interaction to be determined, and potential drug molecules
to be identified.

Barrie Kellam (Professor of Pharmaceutical Medicinal Chemistry,
University of Nottingham 1997-present) first synthesised
fluorescently-tagged small molecule antagonists and agonists to the
adenosine receptor in the School of Pharmacy from 2001-2006 (1,2). The
first ligand synthesised was the antagonist XAC-BY630, based on the
xanthine amine congener conjugated to a BODIPY630/650 fluorescent tag
(fluorophore) (1). This was followed up with a series of agonists based on
5'-N-ethyl carboxamidoadenosine conjugated to BODIPY630/650 (2).
Pharmacological validation of these ligands was carried out in
collaboration with Prof Stephen Hill and Dr Stephen Briddon of the School
of Biomedical Sciences. Hill and Briddon used functional assays and
confocal imaging to confirm interaction of the ligands with the adenosine
receptor and Fluorescence Correlation Spectroscopy (FCS) to quantify
ligand binding in membrane microdomains of single living cells (1-3).
Further research showed that the success of these fluorescently-tagged
ligands depends greatly on the chemical linker used to conjugate the
ligand to the fluorophore, and also the type of fluorophore used. Varying
these parameters had a profound effect on the binding affinity and
efficacy of the ligands. Factors influencing the binding affinity were
evaluated between 2004 and 2006 and were shown to include the length and
physicochemical properties of the linker, site of linkage, and size of
attached fluorophore (3). Continuing research has led to additional GPCRs
being targeted and more selective ligands for the adenosine receptor
family being synthesised: ligands for the 03b2-adrenoceptor family
(therapeutic targets in cardiovascular and respiratory diseases) were
synthesised and characterised in 2010 (4) and highly potent and selective
ligands for the adenosine A1-receptor (involved in a broad range of
signalling responses) and the adenosine A3-receptor (a therapeutic target
for cancer, glaucoma and autoimmune inflammatory disorders) were
synthesised and evaluated in 2011 (5,6).

Initial funding for this new research was provided by the University of
Nottingham and the Wellcome Trust (7,8). The BBSRC funded work on
adenosine receptor binding in 2005 (9), which was subsequently followed up
with a major programme grant from the MRC in 2009 (10). Novartis also
funded a BBSRC CASE studentship in 2008 with Kellam to develop fluorescent
muscarinic receptor ligands for their in house studies.

Details of the impact

Fluorescent ligand technologies developed at the University of Nottingham
have been commercialised through the spin-out company CellAura.
Distributer agreements have made these ligands globally available,
enabling industry to gain new insights into receptor ligand interactions.
In addition, the use of fluorescent ligands allows for faster, more
cost-effective screening studies that negate the need for radio-labelling,
making ligand binding studies safer and reducing the associated
environmental impact.

The initial publication in the Proceedings of the National Academy of
Sciences (1) described for the first time a pharmacologically-validated
fluorescent ligand for a GPCR and the application of FCS to the study of
non-peptide GPCR ligands in membrane micro-domains of single living cells.
This work and the patents (11,12) that were filed before this publication
formed the basis for the establishment of the spin-out company CellAura
Technologies Ltd in 2004. CellAura, based at Nottingham BioCity, began
trading in 2006 and has subsequently received investment funds of £1.8M
(a). Since 2008, CellAura have maintained a workforce up to 7 FT and 4 PT
staff (a).

CellAura currently market 51 products (17 validated ligands and 34
development ligands, www.cellaura.com).
These products are underpinned by the novel ligand development process
patented by the University of Nottingham (11,12), which has been
exclusively licenced to CellAura since 2006. In some cases, ligands have
been developed in collaboration with the University (4,6) via a pipeline
agreement signed in 2008 (a). The unique selling point of these products
is that they are pharmacologically validated and provide applications not
previously available by conventional methods — they can be used on primary
cells (including from diseased tissue), on single living cells, in real
time and in the native environment — providing new information on binding
kinetics and receptor distribution, internalisation and signalling. They
can also be used to perform traditional receptor-ligand binding studies at
comparable cost to using radio-ligands without the need for associated
radioactive licences, infrastructure and safety monitoring, making these
studies more amenable to smaller research companies for whom it is not
practicable to have their own radiation facilities.

To facilitate access to CellAura's products, on-going regional
distributor agreements were arranged with Fisher Scientific (UK and
Scandinavia) and Funakoshi (Japan) in 2009, and worldwide agreements with
Abcam and Sigma-Aldrich in 2011 (a). CellAura also make direct-to-customer
sales in Europe, North America and Australasia, and provide custom ligand
development contracts. Their customers include the major pharmaceutical
companies AstraZeneca, Pfizer, Sanofi Aventis, Amgen and Takeda, and GPCR
biotechnology companies such as Addex and Heptares (a). Novartis
Pharmaceuticals UK Ltd has used CellAura ligands to understand the
mechanism of `tethered' drugs which has led to a new direction in their
research into GPCR drugs. The Global Head of Respiratory at Novartis
confirmed the benefits to the company "Novartis' studies with
fluorescent derivatives of therapeutic compounds have led to the
ground-breaking concept of tethered drugs which Novartis has applied to
better understand the pharmacological mechanism of drug compounds and to
direct new research on longer acting drugs targeting GPCRs. It is
therefore an enabling technology that brings benefits to the
pharmaceutical industry by allowing the pursuit of approaches that were
previously unavailable". (b)

CellAura ligands are also utilised by drug discovery technology
providers. Between 2009 and 2012, CellAura developed a number of custom
fluorescent ligands for CisBio Bioassays, a French-based global drug
discovery assay development company. CisBio adopted 18 `active' ligands
(prepared exclusively for CisBio and not available via the CellAura
catalogue) for use in their proprietary Tag-lite™ GPCR high throughput
screening (HTS) platform (a,c). Promega Corporation, a US-based market
leader in developing and commercialising novel reporter technologies and
assay chemistries for interrogating GPCR signalling, has been using
CellAura ligands since 2011 to successfully validate their innovative
NanoLuc® Luciferase Technology. The combined use of this superior
luciferase technology along with high affinity fluorescent ligands has
also enabled GPCR-ligand interactions to be studied via Bioluminescence
Resonance Energy Transfer (BRET; d). Promega state that "The
fluorescent ligands developed by CellAura have proven to be excellent
tracers for measuring the affinities of various GPCR drugs in the
competitive displacement format, and have helped to validate NanoLuc as
an excellent reporter tag for ligand binding studies using BRET. Data
presented to customers in the pharma industry in 2012 and 2013 have
spurred considerable interest in NanoLuc/BRET for general target
engagement assays". (d)

Since 2008, CellAura have demonstrated that their technology works on a
range of functional screening platforms used in the pharmaceutical
industry, including high content analysis (HCA) imaging platforms.
Demonstration results have been disseminated via poster presentations at
various scientific meetings (e) and used in marketing materials. CellAura
was voted `best new technology' at the European Laboratory Robotics
Interest Group (ELRIG)/ SBS Drug Discovery meeting in 2008. Fluorescence
techniques such as HCA rely on high resolution fluorescence image capture
meaning that these techniques are not as fast as traditional methods for
receptor ligand binding, and are in general being used to provide
additional information alongside rather than replacing radio-ligands in
HTS. In 2012 CellAura established a successful collaboration with BMG
Labtech (a German-based global developer and manufacturer of microplate
readers) that enables CellAura's ligands to be utilised on a conventional
(non-imaging) PheraStar fluorescence plate reader. Ligand binding assays
analogous to radio-ligand studies can be performed on the PheraStar with
inherent safety advantages. The PheraStar also has time saving advantages
over HCA (all inclusive 96 well plate assay time of <10 minutes,
compared to approx. 1 hour) and gives comparable binding data, making this
technique more amenable for HTS. Technical notes outlining this technique
are made available via both the CellAura and BMG Labtech websites (f). In
addition, a PheraStar plate reader costs much less than a HCA imaging
platform (approximately £70K compared to £450K (i)). Together, these
fluorescent ligand binding techniques provide improved methods for
investigating ligand interactions with GPCRs (discussed by Comley in the
industrial publication, Drug Discovery World Spring 2009, 32-50)
and time and cost benefits (particularly when using specific screening
platforms) for drug discovery, important factors when promoting new
technology to a pharmaceutical industry that has undergone major
realignment in recent years.

In addition to the use of fluorescent ligands in drug discovery, CellAura
have diversified the impact of the technology to include live cell
sorting. This new patented technology (g), filed by CellAura in 2007,
describes the novel use of fluorescent ligands as alternatives to
antibodies for fluorescence activated cell sorting. The method described
can be used to easily sort cells expressing specific receptors when
generating recombinant clonal cell lines. CellAura ligands have been used
by the German SME inSCREENex GmbH since 2009 to establish cell lines for
drug discovery. Their Managing Director confirmed the benefits of using
CellAura ligands "The performance [of the ligands] was great and
superior to any of the antibodies we used in parallel. By using the
ligands we were able to reduce the time to analyse our cell lines
significantly. Further, the application of the ligands allowed us to
screen a high number of novel cell lines for optimal expression. This is
not possible with conventional methods like Western Blotting." (h)

In summary, commercialisation of fluorescent ligands through CellAura has
opened up new and improved methods for industrial drug discovery, and a
number of distributer agreements have made these ligands globally
available.